Recent epidemiological studies have strongly linked the common consumption of coffee and other caffeinated beverages to a significantly reduced risk of developing Parkinson's disease (PD), raising the possibility of neuroprotection by caffeine. Many CNS effects of caffeine have been attributed to its antagonistic actions on the A2A subtype of adenosine receptor (A2AR). We have recently found that caffeine and chlorostyryl-caffeine (a more specific A2A antagonist) as well as genetic inactivation of the A2AR all protect against MPTP neurotoxicity in mice. Furthermore, we have observed that A2AR blockade or A2A deficiency can reduce the levels of the toxic MPTP metabolite, MPP+ in vivo. Based on the convergence of epidemiological and molecular data, we hypothesize that caffeine attenuates neurotoxin-induced dopaminergic neuron degeneration through a novel A2A-dependent mechanism. We propose (in response to PA 99-054) to systematically characterize caffeine-induced neuroprotection and its mechanism in the MPTP model of PD using complementary pharmacological and genetic approaches to A2AR inactivation. Specific Aim 1 will investigate the effects of caffeine on MPTP-induced neurotoxicity by analyzing the death as well as dysfunction of dopaminergic nigrostriatal neurons (i.e., dopaminergic neuron loss in the substantia nigra, as well as depletion of dopamine content and re-uptake sites in the striatum). Caffeine's potency and time course of protection against these MPTP-induced nigrostriatal lesions will be established. Specific Aim 2 will determine caffeine's initial mechanism of action in the attenuation of MPTP toxicity. Pharmacological consideration of caffeine's known molecular targets as candidate mediators of neuroprotection will be pursued by comparing the effects of specific inhibitors of A1 receptors, A2 receptors, GABAA receptors and phosphodiesterase activity in the MPTP model. A more definitive demonstration of A2AR involvement will be pursued by comparing the effects of caffeine in wild-type and A2AR knockout mice. Specific Aim 3 will explore two plausible mechanisms of A2AR-mediated neuroprotection by caffeine. We will examine MPTP metabolism in vivo and monoamine oxidase activity in vitro to pursue the possibility that altered neurotoxin metabolism contributes to the neuroprotective effects of caffeine. Lastly, based on evidence that A2AR blockade reduces striatal glutamate and dopamine release, we will assess the ability of caffeine and A2AR inactivation to reduce the potentially toxic extracellular levels of these neurotransmitters induced by MPTP in vivo. Together these experiments stand to establish a novel neuroprotective role for A2AR inactivation in the MPTP model of PD, as well as a potential mechanism by which caffeine may reduce the risk of developing PD. As specific A2A receptor antagonists are now being introduced in PD clinical trials for their acute motor benefits, an understanding of their effects on the underlying neurodegenerative process in PD may be of considerable therapeutic, as well as epidemiological, significance.